| In this study,the young stems of Pueraria thomsonii Benth were used as experimental materials to study the effects of explant types,culture conditions,hormone levels,sub-generation cycles,and number of sub-generations on the induction of loose callus of Pueraria thomsonii Benth.A suspension cell mass culture system of Pueraria thomsonii Benth was established in the wound tissue.The effects of high salt and low osmotic stress treatments on the growth of Pueraria thomsonii Benth suspension cells and the content of secondary metabolites(total flavonoids,soluble sugars,and soluble proteins)were studied.The main results are as follows:1.Loose callus induction of Pueraria thomsonii Benth.Taking the young plants of Pueraria thomsonii Benth as experimental materials,the effects of different explant,culture conditions,subculture cycles,hormone levels,and the number of subcultures on the induction of loose callus of Pueraria thomsonii Benth were studied.The results showed that the suitable explant site for the callus induction of Pueraria thomsonii Benth is the stem,and the conditions suitable for the callus culture of Pueraria thomsonii Benth are shading for the first 20 days and light for the next 10 days.The best number of subcultures for Pueraria thomsonii Benth is the fourth.Second,the best subgeneration cycle is 7 days,and the hormone level condition is0.4 mg·L-1IBA+0.5mg·L-16-BA.2.Establishment of Pueraria thomsonii Benth suspension cell mass culture system.Using loose callus subcultured for 3-4 times as materials,the basic medium,sucrose concentration,three physical treatment methods(crushing,stirring,crushing and stirring)and enzyme treatment on the suspension cells of Pueraria thomsonii Benth were studied.The influence of fresh weight and total flavonoids.The results showed that MS medium is more suitable for the growth and dispersion of Pueraria thomsonii Benth suspension cell clusters.When the sucrose concentration is30 g·L-1,it is most suitable for the growth of Pueraria thomsonii Benth suspended cell clusters and the accumulation of total flavonoids.Conducive to the dispersion of the suspension cell clusters of Pueraria thomsonii Benth.3.The effect of low nutrient stress on the fresh weight,soluble sugar,soluble protein and total flavonoids accumulated inside and outside the suspension cells of Pueraria thomsonii Benth.The results showed that under this stress,the fresh weight of the suspension cells of Pueraria thomsonii Benth rose first and then decreased.The soluble sugar in the cells basically decreased first and then increased,but their content was lower than the control content of the same period.The soluble sugar in the cells The protein content showed a downward trend.In the early period(3-5 days),its content was higher than the same period control,and the soluble protein content of Pueraria thomsonii Benth suspension cells was lower than the same period control in the later period(7-11 days).The content of total flavonoids in the suspension cells of Pueraria thomsonii Benth showed a downward trend,and the content of total flavonoids in the suspension showed a trend of first decline and then rise.4.The effect of salt stress on the fresh weight,soluble sugar,soluble protein and total flavonoids accumulated inside and outside the suspension cells of Pueraria thomsonii Benth.The results showed that under this stress,as the salt concentration increased,the fresh weight of the suspension cells of Pueraria thomsonii Benth showed a downward trend.When the salt concentration was 100mmol·L-1and 150mmol·L-1,the fresh weight of the cells was equal When the salt concentration is 50mmol·L-1,the cell fresh weight is higher than the control content on the3-5 days,and lower than the control content on the 7-11 days.Under different salt stress treatments,the soluble sugar content was lower than that of the control at the same time.The soluble protein content in the suspension cells of Pueraria thomsonii Benth also showed a downward trend with the increase of salt concentration,and its content was basically lower than that of the control at the same time.The content of total flavonoids in suspension cells increases with the increase of salt concentration.When the salt concentration is 150mmol·L-1,the total flavonoids content in suspension cells is the highest on the 5th day,and the total flavonoids content in the cells is the highest when the salt concentration is 150mmol·L-1.The content of total flavonoids in the suspension is the highest.5.The effect of low osmotic stress on the fresh weight,soluble sugar,soluble protein,and total flavonoid content accumulation in suspension cells of Pueraria thomsonii Benth.The results showed that as the concentration of polyethylene glycol increased,the fresh weight of the suspension cells basically showed a downward trend.When the concentration of polyethylene glycol was 5%,the fresh weight of the suspension cells reached the maximum on the 11th day.Under different concentrations of polyethylene glycol stress,the soluble sugar content in the suspension cells of Pueraria thomsonii Benth was lower than the soluble sugar content in the control during the same period.Under different concentrations of PEG stress,the soluble protein content in the suspension cells of Pueraria thomsonii Benth was higher than the soluble protein content in the control during the same period.When the PEG concentration was 5%suspension culture to the 9th day,the soluble protein content and the soluble sugar content in the cells were both reaches the maximum value.The content of total flavonoids in the suspension cells of Pueraria thomsonii Benth basically decreased first and then increased with the increase of PEG concentration.The total flavonoid content in the suspension basically increased with the increase of PEG concentration.When the PEG concentration was 10%suspension culture to the 5th day,the total flavonoid content in the suspension cells of Pueraria thomsonii Benth and the total flavonoid content in the suspension reached the maximum value. |
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